1. Field of the Invention
The invention relates to mammalian kinase ligands, substrates and modulators. In particular, the invention relates to polypeptides, polypeptide compositions and polynucleotides that encode polypeptides that are ligands, substrates, and/or modulators of PKA. The invention also relates to polyligands that are homopolyligands or heteropolyligands that modulate PKA activity. The invention also relates to ligands and polyligands tethered to a subcellular location.
2. Background of the Invention
Kinases are enzymes that catalyze the addition of phosphate to a molecule. The addition of phosphate by a kinase is called phosphorylation. When the kinase substrate is a protein molecule, the amino acids commonly phosphorylated are serine, threonine and tyrosine. Phosphatases are enzymes that remove phosphate from a molecule. The removal of phosphate is called dephosphorylation. Kinases and phosphatases often represent competing forces within a cell to transmit, attenuate, or otherwise modulate cellular signals and cellular control mechanisms. Kinases and phosphatases have both overlapping and unique natural substrates. Cellular signals and control mechanisms, as regulated by kinases, phosphatases, and their natural substrates are a target of research tool design and drug design.
Mammalian cyclic AMP-dependent protein kinase is also known as Protein Kinase A or PKA. PKA can phosphorylate serine and threonine residues. The enzymatic activity, activation and regulation of PKA have been studied. Many cellular and peptide substrates of PKA have been identified (See for example, Banke et al. 2000 J Neurosci 20:89-102; Bhave et al. 2002 Neuron 35:721-731; Blumenthol et al. 1986 J Biol Chem 261:8140-45; Boo 2006 Exper. Molec. Medicine 38:63-71; Bunemann et al. 1999 J Biol Chem 274:33851-54; Butt et al. 1994 J Biol Chem 269:14509-17; Chang et al. 1993 J Biol Chem 268:11304-11; Chen et al. 2005 J Biol Chem 280:31347-52; Cheng et al. 1986 J Biol Chem 261:989-992; Chheda et al. 2001 Nature Cell Biol 3:331-338; Collins et al. 2000 Biochem J 345:673-680; Day et al. 1989 J Biol Chem 264:431-436; D'Sousa et al. 2005 J Biol Chem 280:26233-40; Feramisco et al. 1978 J Biol Chem 253:8968-71; Ferris et al. 1991 Proc Natl Acad Sci USA 88:2232-35; Giordano et al. 2005 J Neurochem 94:808-818; Glass et al. 1989 J Biol Chem 264:8802-8810; Hall et al. 2006 Biochemistry 45:3448-3459; Harada et al. 1999 Molecular Cell 3:413-422; Homer et al. 2005 J Biol Chem 280:28241-50; Hsieh-Wilson et al. 2003 J Biol Chem 278-1186-94; Jay et al. 2004 Mol Cell Biochem 260:49-53; Ji et al. 2003 J Biol Chem 278:25063-71; Kameyama et al. 1998 Neuron 21:1163-75; Kemp et al. 1977 J Biol Chem 252:4888-4894; Kemp et al. 1978 J Biol Chem 253:5147-54; Lambrechts et al. 2000 J Biol Chem 275:36143-36151; Liu et al. 2004 J Biol Chem 279:50078-88; Lu et al. 2002 Virology 300:20-30; Maller et al. 1978 Proc Natl Acad Sci USA 75:248-251; Meetei et al. 2002 Biochemistry 41:185-195; Pi et al. 2002 Circ Res 90:649-656; Quilliam et al. 1991 J Immunol 147:1628-1635; Sakthivel et al. 2005 J Biol Chem 280:703-714; Schmidt et al. 2002 J Virol 76:1033-42; Scott et al. 1985 Proc Natl Acad Sci USA 82:4397-83; Scott et al. 1986 proc Natl Acad Sci USA 83:1613-16; Sette et al. 1996 J Biol Chem 271:16526-34; Snyder et al. 2004 J Biol Chem 279:45753-58; Taylor 1982 J Biol Chem 257:6056-6063; Walsh et al. 1994 FASEB J 8:1227-1236; Wecker et al. 2003 Neurochem Res 28:431-436; Wright et al. 1981 Proc Natl Acad Sci USA 78:6048-6050; Wullrich et al. 1993 J Biol Chem 268:23208-14; Xu et al. 1996 J Biol Chem 271:9313-19; Yamamoto et al. 2002 Biochem Pharmacol 64:375-383; Zhou et al. 2003 J Biol Chem 278:35651-59). Natural and synthetic polypeptides have been studied to examine PKA substrate specificity. While polypeptides and variants thereof have been studied as individual PKA substrates or ligands, mixed ligands linked together as polyligands that modulate PKA activity have not been demonstrated before this invention. An aspect of the invention is to provide novel, modular, inhibitors of PKA activity by modifying one or more natural substrates either by truncation or by amino acid substitution. A further aspect of the invention is the subcellular localization of a PKA inhibitor, ligand, or polyligand by linking to a subcellular localization signal.
Disruption of PKA binding protein interactions in sperm has been disclosed. See, for example, U.S. Pat. Nos. 6,011,013 and 6,451,528.
Further, there are several small molecule agents known in the art and used experimentally, such as H-89, HA-1004, H-7, H-8, HA-100 and staurosporine, that inhibit PKA activity.
Design and synthesis of polypeptide ligands that modulate calcium/calmodulin-dependent protein kinase and that localize to the cardiac sarco(endo)plasmic reticulum was performed by Ji et al. (J Biol Chem (2003) 278:25063-71). Ji et al. accomplished this by generating expression constructs that localized calcium/calmodulin-dependent protein kinase inhibitory polypeptide ligands to the sarcoplasmic reticulum by fusing a sarcoplasmic reticulum localization signal derived from phospholamban to a polypeptide ligand. See also U.S. Pat. No. 7,071,295.